OBJECTIVE: To analyze the clinical phenotype and pathogenic mechanism of the ARSL gene variant in a fetus with nasal bone aplasia. METHODS: A 34-year-old pregnant woman who attended Quzhou Maternal and Child Health Care Hospital on January 3, 2023 was selected as the study subject. Whole exome sequencing (WES) was performed on the fetus. Bioinformatics analysis was carried out to identify and prioritize candidate gene variants, followed by Sanger sequencing for familial validation. A mutant plasmid expression vector was constructed and subsequently transfected into HEK293T cells to preliminarily investigate the pathogenetic mechanism of the identified variant. Additionally, a comprehensive review of literature was conducted to systematically summarize the associated clinical phenotypes. This study was approved by the Medical Ethics Committee of Quzhou Maternal and Child Health Care Hospital (Ethics No.: KY-2023-11). RESULTS: WES revealed that the fetus harbored a c.827del (p.L276Rfs*48) variant of the ARSL gene, for which its mother was heterozygous. Based on the guidelines from the American College of Medical Genetics and Genomics (ACMG), the variant was classified as pathogenic(PVS1+PM2_Supporting). In vitro cellular function studies demonstrated that this variant can result in a substantial decrease in the expression of mutant mRNA, thereby preventing the production of normal ARSL protein. Clinical phenotypes resulting from ARSL gene variants exhibited considerable diversity, with nasal hypoplasia being the most common manifestation. CONCLUSION The c.827del (p.L276Rfs*48) variant of the ARSL gene can lead to degradation of mRNA via the nonsense-mediated mRNA decay pathway, resulting in reduced levels of ARSL protein. The pathogenetic mechanism underlying the ARSL gene variant may be associated with its haploinsufficiency effect.
Humans ; Female ; Pregnancy ; Adult ; Frameshift Mutation ; HEK293 Cells ; Nasal Bone/abnormalities* ; Fetus/abnormalities* ; Exome Sequencing

OBJECTIVE: To analyze the functional impact of a rare heterozygous variant of SOS1 gene (c.283G>A, p.E95K) identified in a fetus with cervical cystic hygroma and to explore its association with the disease phenotype. METHODS: A pedigree analysis was carried out to evaluate the co-segregation of the variant with the disease phenotype. Bioinformatic tools were employed to assess the conservation, protein structure and stability. Functional validation was conducted on HEK293T cells using fluorescence quantitative reverse transcription-PCR and Western blotting to measure the expression of SOS1 and phosphorylation levels of extracellular regulated protein kinases (ERK) and c-Jun N-terminal kinase. A literature review of previously reported disease-associated SOS1 variants was also carried out. This study has been approved by the Medical Ethics Committee of West China Second University Hospital, Sichuan University (Ethics No.: 201940). RESULTS: The variant was inherited from the husband of the woman with distinctive facial features and has co-segregated with the phenotype. Bioinformatics analysis indicated that the variant is located in a highly conserved region, and that p.E95K could disrupt key amino acid interactions and protein stability. Multiple bioinformatic predictions consistently suggested the pathogenicity of this variant. Functional assays demonstrated reduced SOS1 protein expression and decreased ERK phosphorylation. CONCLUSION This study has revealed the functional impact of the SOS1 c.283G>A (p.E95K) variant, suggesting that it may contribute to the developmental phenotypes through a haploinsufficiency mechanism.
Humans ; SOS1 Protein/chemistry* ; Female ; HEK293 Cells ; Male ; Pedigree ; Phenotype ; Adult

BACKGROUND: P16 inactivation is frequently accompanied by telomerase reverse transcriptase ( TERT ) amplification in human cancer genomes. P16 inactivation by DNA methylation often occurs automatically during immortalization of normal cells by TERT . However, direct evidence remains to be obtained to support the causal effect of epigenetic changes, such as P16 methylation, on cancer development. This study aimed to provide experimental evidence that P16 methylation directly drives cancer development. METHODS: A zinc finger protein-based P16 -specific DNA methyltransferase (P16-Dnmt) vector containing a "Tet-On" switch was used to induce extensive methylation of P16 CpG islands in normal human fibroblast CCD-18Co cells. Battery assays were used to evaluate cell immortalization and transformation throughout their lifespan. Cell subcloning and DNA barcoding were used to track the diversity of cell evolution. RESULTS: Leaking P16-Dnmt expression (without doxycycline-induction) could specifically inactivate P16 expression by DNA methylation. P16 methylation only promoted proliferation and prolonged lifespan but did not induce immortalization of CCD-18Co cells. Notably, cell immortalization, loss of contact inhibition, and anchorage-independent growth were always prevalent in P16-Dnmt&TERT cells, indicating cell transformation. In contrast, almost all TERT cells died in the replicative crisis. Only a few TERT cells recovered from the crisis, in which spontaneous P16 inactivation by DNA methylation occurred. Furthermore, the subclone formation capacity of P16-Dnmt&TERT cells was two-fold that of TERT cells. DNA barcoding analysis showed that the diversity of the P16-Dnmt&TERT cell population was much greater than that of the TERT cell population. CONCLUSION P16 methylation drives TERT -mediated immortalization and transformation of normal human cells that may contribute to cancer development.
Humans ; Telomerase/genetics* ; DNA Methylation/physiology* ; Fibroblasts/cytology* ; Cyclin-Dependent Kinase Inhibitor p16/metabolism* ; Cell Line ; Cell Transformation, Neoplastic/genetics*

BACKGROUND: Chronic kidney disease (CKD) is associated with common pathophysiological processes, such as inflammation and fibrosis, in both the heart and the kidney. However, the underlying molecular mechanisms that drive these processes are not yet fully understood. Therefore, this study focused on the molecular mechanism of heart and kidney injury in CKD. METHODS: We generated an microRNA (miR)-26a knockout (KO) mouse model to investigate the role of miR-26a in angiotensin (Ang)-II-induced cardiac and renal injury. We performed Ang-II modeling in wild type (WT) mice and miR-26a KO mice, with six mice in each group. In addition, Ang-II-treated AC16 cells and HK2 cells were used as in vitro models of cardiac and renal injury in the context of CKD. Histological staining, immunohistochemistry, quantitative real-time polymerase chain reaction (PCR), and Western blotting were applied to study the regulation of miR-26a on Ang-II-induced cardiac and renal injury. Immunofluorescence reporter assays were used to detect downstream genes of miR-26a, and immunoprecipitation was employed to identify the interacting protein of LIM and senescent cell antigen-like domain 1 (LIMS1). We also used an adeno-associated virus (AAV) to supplement LIMS1 and explored the specific regulatory mechanism of miR-26a on Ang-II-induced cardiac and renal injury. Dunnett's multiple comparison and t -test were used to analyze the data. RESULTS: Compared with the control mice, miR-26a expression was significantly downregulated in both the kidney and the heart after Ang-II infusion. Our study identified LIMS1 as a novel target gene of miR-26a in both heart and kidney tissues. Downregulation of miR-26a activated the LIMS1/integrin-linked kinase (ILK) signaling pathway in the heart and kidney, which represents a common molecular mechanism underlying inflammation and fibrosis in heart and kidney tissues during CKD. Furthermore, knockout of miR-26a worsened inflammation and fibrosis in the heart and kidney by inhibiting the LIMS1/ILK signaling pathway; on the contrary, supplementation with exogenous miR-26a reversed all these changes. CONCLUSIONS Our findings suggest that miR-26a could be a promising therapeutic target for the treatment of cardiorenal injury in CKD. This is attributed to its ability to regulate the LIMS1/ILK signaling pathway, which represents a common molecular mechanism in both heart and kidney tissues.
Animals ; MicroRNAs/metabolism* ; Angiotensin II/toxicity* ; Mice ; Renal Insufficiency, Chronic/chemically induced* ; Mice, Knockout ; Disease Models, Animal ; Male ; Signal Transduction/genetics* ; LIM Domain Proteins/genetics* ; Mice, Inbred C57BL ; Cell Line ; Humans

BACKGROUND: The transcription factor POU2F1 regulates the expression levels of microRNAs in neoplasia. However, the miR-29b1/a cluster modulated by POU2F1 in gastric cancer (GC) remains unknown. METHODS: Gene expression in GC cells was evaluated using reverse-transcription polymerase chain reaction (PCR), western blotting, immunohistochemistry, and RNA in situ hybridization. Co-immunoprecipitation was performed to evaluate protein interactions. Transwell migration and invasion assays were performed to investigate the biological behavior of GC cells. MiR-29b1/a cluster promoter analysis and luciferase activity assay for the 3'-UTR study were performed in GC cells. In vivo tumor metastasis was evaluated in nude mice. RESULTS: POU2F1 is overexpressed in GC cell lines and binds to the miR-29b1/a cluster promoter. POU2F1 is upregulated, whereas mature miR-29b-3p and miR-29a-3p are downregulated in GC tissues. POU2F1 promotes GC metastasis by inhibiting miR-29b-3p or miR-29a-3p expression in vitro and in vivo . Furthermore, PIK3R1 and/or PIK3R3 are direct targets of miR-29b-3p and/or miR-29a-3p , and the ectopic expression of PIK3R1 or PIK3R3 reverses the suppressive effect of mature miR-29b-3p and/or miR-29a-3p on GC cell metastasis and invasion. Additionally, the interaction of PIK3R1 with PIK3R3 promotes migration and invasion, and miR-29b-3p , miR-29a-3p , PIK3R1 , and PIK3R3 regulate migration and invasion via the phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/Akt/mTOR) pathway in GC cells. In addition, POU2F1 , PIK3R1 , and PIK3R3 expression levels negatively correlated with miR-29b-3p and miR-29a-3p expression levels in GC tissue samples. CONCLUSIONS The POU2F1 - miR-29b-3p / miR-29a-3p-PIK3R1 / PIK3R1 signaling axis regulates tumor progression and may be a promising therapeutic target for GC.
MicroRNAs/metabolism* ; Humans ; Stomach Neoplasms/pathology* ; Cell Line, Tumor ; Cell Movement/physiology* ; Phosphatidylinositol 3-Kinases/metabolism* ; Animals ; Mice ; Octamer Transcription Factor-1/metabolism* ; Mice, Nude ; Class Ia Phosphatidylinositol 3-Kinase/metabolism* ; Neoplasm Invasiveness ; Gene Expression Regulation, Neoplastic/genetics* ; Male ; Immunohistochemistry ; Female

BACKGROUND: Oral squamous cell carcinoma (OSCC) is a prevalent type of cancer with a high mortality rate in its late stages. One of the major challenges in OSCC treatment is the resistance to epidermal growth factor receptor (EGFR) inhibitors. Therefore, it is imperative to elucidate the mechanism underlying drug resistance and develop appropriate precision therapy strategies to enhance clinical efficacy. METHODS: To evaluate the efficacy of the combination of the Ca 2+ /calmodulin-dependent protein kinase II (CAMK2) inhibitor KN93 and EGFR inhibitors, we performed in vitro and in vivo experiments using two FAT atypical cadherin 1 ( FAT1 )-deficient (SCC9 and SCC25) and two FAT1 wild-type (SCC47 and HN12) OSCC cell lines. We assessed the effects of EGFR inhibitors (afatinib or cetuximab), KN93, or their combination on the malignant phenotype of OSCC in vivo and in vitro . The alterations in protein expression levels of members of the EGFR signaling pathway and SRY-box transcription factor 2 (SOX2) were analyzed. Changes in the yes-associated protein 1 (YAP1) protein were characterized. Moreover, we analyzed mitochondrial dysfunction. Besides, the effects of combination therapy on mitochondrial dynamics were also evaluated. RESULTS: OSCC with FAT1 mutations exhibited resistance to EGFR inhibitors treatment. The combination of KN93 and EGFR inhibitors significantly inhibited the proliferation, survival, and migration of FAT1 -mutated OSCC cells and suppressed tumor growth in vivo . Mechanistically, combination therapy enhanced the therapeutic sensitivity of FAT1 -mutated OSCC cells to EGFR inhibitors by modulating the EGFR pathway and downregulated tumor stemness-related proteins. Furthermore, combination therapy induced reactive oxygen species (ROS)-mediated mitochondrial dysfunction and disrupted mitochondrial dynamics, ultimately resulting in tumor suppression. CONCLUSION Combination therapy with EGFR inhibitors and KN93 could be a novel precision therapeutic strategy and a potential clinical solution for EGFR-resistant OSCC patients with FAT1 mutations.
Humans ; ErbB Receptors/metabolism* ; Mouth Neoplasms/metabolism* ; Cell Line, Tumor ; Animals ; Drug Resistance, Neoplasm/genetics* ; Cadherins/metabolism* ; Carcinoma, Squamous Cell/metabolism* ; Mice ; Mutation/genetics* ; Mice, Nude ; Protein Kinase Inhibitors/therapeutic use* ; Cetuximab/pharmacology* ; Afatinib/therapeutic use* ; Cell Proliferation/drug effects* ; Signal Transduction/drug effects*

BACKGROUND: T-cell lymphoblastic lymphoma/acute lymphoblastic leukemia (T-LBL/ALL) is an aggressive form of hematological malignancy associated with poor prognosis in adult patients. Histone deacetylases (HDACs) are aberrantly expressed in T-LBL/ALL and are considered potential therapeutic targets. Here, we investigated the antitumor effect of a novel HDAC inhibitor, chidamide, on T-LBL/ALL. METHODS: HDAC1, HDAC2 and HDAC3 levels in T-LBL/ALL cell lines and patient samples were compared with those in normal controls. Flow cytometry, transmission electron microscopy, and lactate dehydrogenase release assays were conducted in Jurkat and MOLT-4 cells to assess apoptosis and pyroptosis. A specific forkhead box O1 (FOXO1) inhibitor was used to rescue pyroptosis and upregulated gasdermin E (GSDME) expression caused by chidamide treatment. The role of the FOXO1 transcription factor was evaluated by dual-luciferase reporter and chromatin immunoprecipitation assays. The efficacy of chidamide in vivo was evaluated in a xenograft mouse. RESULTS: The expression of HDAC1, HDAC2 and HDAC3 was significantly upregulated in T-LBL/ALL. Cell viability was obviously inhibited after chidamide treatment. Pyroptosis, characterized by cell swelling, pore formation on the plasma membrane and lactate dehydrogenase leakage, was identified as a new mechanism of chidamide treatment. Chidamide triggered pyroptosis through caspase 3 activation and GSDME transcriptional upregulation. Chromatin immunoprecipitation assays confirmed that chidamide led to the increased transcription of GSDME through a more relaxed chromatin structure at the promoter and the upregulation of FOXO1 expression. Moreover, we identified the therapeutic effect of chidamide in vivo . CONCLUSIONS This study suggested that chidamide exerts an antitumor effect on T-LBL/ALL and promotes a more inflammatory form of cell death via the FOXO1/GSDME axis, which provides a novel choice of targeted therapy for patients with T-LBL/ALL.
Humans ; Pyroptosis/drug effects* ; Forkhead Box Protein O1/genetics* ; Aminopyridines/pharmacology* ; Animals ; Mice ; Benzamides/pharmacology* ; Cell Line, Tumor ; Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/drug therapy* ; Phosphate-Binding Proteins/metabolism* ; Histone Deacetylase Inhibitors/pharmacology* ; Jurkat Cells ; Histone Deacetylases/metabolism* ; Apoptosis/drug effects* ; Gasdermins

BACKGROUND: Growing evidence suggests that long non-coding RNAs (lncRNAs) exert pivotal roles in fostering chemoresistance across diverse tumors. Nevertheless, the precise involvement of lncRNAs in modulating chemoresistance within the context of gallbladder cancer (GBC) remains obscure. This study aimed to uncover how lncRNAs regulate chemoresistance in gallbladder cancer, offering potential targets to overcome drug resistance. METHODS: To elucidate the relationship between gemcitabine sensitivity and small nucleolar RNA host gene 1 ( SNHG1 ) expression, we utilized publicly available GBC databases, GBC tissues from Renji Hospital collected between January 2017 and December 2019, as well as GBC cell lines. The assessment of SNHG1, miR-23b-3p, and phosphatase and tensin homolog (PTEN) expression was performed using in situ  hybridization, quantitative real-time polymerase chain reaction, and western blotting. The cell counting kit-8 (CCK-8) assay was used to quantify the cell viability. Furthermore, a GBC xenograft model was employed to evaluate the impact of SNHG1 on the therapeutic efficacy of gemcitabine. Receiver operating characteristic (ROC) curve analyses were executed to assess the specificity and sensitivity of SNHG1. RESULTS: Our analyses revealed an inverse correlation between the lncRNA SNHG1 and gemcitabine resistance across genomics of drug sensitivity in cancer (GDSC) and Gene Expression Omnibus (GEO) datasets, GBC cell lines, and patients. Gain-of-function investigations underscored that SNHG1 heightened the gemcitabine sensitivity of GBC cells in both in vitro and in vivo  settings. Mechanistic explorations illuminated that SNHG1 could activate PTEN -a commonly suppressed tumor suppressor gene in cancers-thereby curbing the development of gemcitabine resistance in GBC cells. Notably, microRNA (miRNA) target prediction algorithms unveiled the presence of miR-23b-3p binding sites within SNHG1 and the 3'-untranslated region (UTR) of PTEN . Moreover, SNHG1 acted as a sponge for miR-23b-3p, competitively binding to the 3'-UTR of PTEN , thereby amplifying PTEN expression and heightening the susceptibility of GBC cells to gemcitabine. CONCLUSION The SNHG1/miR-23b-3p/PTEN axis emerges as a pivotal regulator of gemcitabine sensitivity in GBC cells, holding potential as a promising therapeutic target for managing GBC patients.
Humans ; Deoxycytidine/pharmacology* ; PTEN Phosphohydrolase/genetics* ; Gemcitabine ; RNA, Long Noncoding/metabolism* ; MicroRNAs/genetics* ; Gallbladder Neoplasms/genetics* ; Cell Line, Tumor ; Animals ; Mice ; Drug Resistance, Neoplasm/genetics* ; Mice, Nude ; Antimetabolites, Antineoplastic ; Gene Expression Regulation, Neoplastic

BACKGROUND: Enzalutamide, a second-generation androgen receptor (AR) pathway inhibitor, is widely used in the treatment of castration-resistant prostate cancer. However, after a period of enzalutamide treatment, patients inevitably develop drug resistance. In this study, we characterized leucine-rich repeated G-protein-coupled receptor 5 (LGR5) and explored its potential therapeutic value in prostate cancer. METHODS: A total of 142 pairs of tumor and adjacent formalin-fixed paraf-fin-embedded tissue samples from patients with prostate cancer were collected from the Pathology Department at Sun Yat-sen Memorial Hos-pital. LGR5 was screened by sequencing data of enzalutamide-resistant cell lines combined with sequencing data of lesions with different Gleason scores from the same patients. The biological function of LGR5 and its effect on enzalutamide resistance were investigated in vitro and in vivo . Glutathione-S-transferase (GST) pull-down, coimmunoprecipitation, Western blotting, and immunofluorescence assays were used to explore the specific binding mechanism of LGR5 and related pathway changes. RESULTS: LGR5 was significantly upregulated in prostate cancer and negatively correlated with poor patient prognosis. Overexpression of LGR5 promoted the malignant progression of prostate cancer and reduced sensitivity to enzalutamide in vitro and in vivo . LGR5 promoted the phosphorylation of glycogen synthase kinase-3β (GSK-3β) by binding heat shock protein 90,000 alpha B1 (HSP90AB1) and mediated the activation of the Wingless/integrated (WNT)/β-catenin signaling pathway. The increased β-catenin in the cytoplasm entered the nucleus and bound to the nuclear AR, promoting the transcription level of AR, which led to the enhanced tolerance of prostate cancer to enzalutamide. Reducing HSP90AB1 binding to LGR5 significantly enhanced sensitivity to enzalutamide. CONCLUSIONS LGR5 directly binds to HSP90AB1 and mediates GSK-3β phosphorylation, promoting AR expression by regulating the WNT/β-catenin signaling pathway, thereby conferring resistance to enzalutamide treatment in prostate cancer.
Male ; Humans ; Phenylthiohydantoin/pharmacology* ; Benzamides ; Receptors, G-Protein-Coupled/genetics* ; Nitriles ; Cell Line, Tumor ; HSP90 Heat-Shock Proteins/metabolism* ; Drug Resistance, Neoplasm/genetics* ; Prostatic Neoplasms/drug therapy* ; beta Catenin/metabolism* ; Receptors, Androgen/genetics* ; Animals ; Mice ; Wnt Signaling Pathway/physiology*

BACKGROUND: Immunosuppression is closely related to the pathogenesis of sepsis, but the underlying mechanisms have not yet been fully elucidated. In this study, we aimed to examine the role of the Sterile Alpha Motif, Src Homology 3 domain and nuclear localization signal 1 (SAMSN1) in sepsis and elucidate its potential molecular mechanism in sepsis induced immunosuppression. METHODS: RNA sequencing databases were used to validate SAMSN1 expression in sepsis. The impact of SAMSN1 on sepsis was verified using gene knockout mice. Flow cytometry was employed to delineate how SAMSN1 affects immunity in sepsis, focusing on immune cell types and T cell functions. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated gene editing in RAW264.7 macrophages enabled interrogation of SAMSN1 's regulatory effects on essential macrophage functions, including cell proliferation and phagocytic capacity. The mechanism of SAMSN1 in the interaction between macrophages and T cells was investigated using the RAW264.7 cell line and primary cell lines. RESULTS: SAMSN1 expression was significantly increased in patients with sepsis and was positively correlated with sepsis mortality. Genetic deletion of Samsn1 in murine sepsis model improved T cell survival, elevated T cell cytolytic activity, and activated T cell signaling transduction. Concurrently, Samsn1 knockout augmented macrophage proliferation capacity and phagocytic efficiency. In macrophage, SAMSN1 binds to Kelch-like epichlorohydrin-associated protein 1 (KEAP1), causing nuclear factor erythroid 2-related factor 2 (NRF2) to dissociate from the KEAP1-NRF2 complex and translocate into the nucleus. This promotes the transcription of the coinhibitory molecules CD48/CD86/carcinoembryonic antigen related cell adhesion molecule 1 (CEACAM1), which bind to their corresponding receptors natural killer cell receptor 2B4/CD152/T cell immunoglobulin and mucin domain-containing protein 3 (TIM3) on the surface of T cells, inducing T-cell exhaustion. CONCLUSIONS SAMSN1 deletion augmented adaptive T cell immunity and macrophage phagocytic-proliferative dual function. Furthermore, it mediates the KEAP1-NRF2 axis, which affects the expression of coinhibitory molecules on macrophages, leading to T-cell exhaustion. This novel immunosuppression mechanism potentially provides a candidate molecular target for sepsis immunotherapy.
Animals ; NF-E2-Related Factor 2/metabolism* ; Mice ; Macrophages/immunology* ; Sepsis/metabolism* ; Kelch-Like ECH-Associated Protein 1/genetics* ; T-Lymphocytes/immunology* ; Humans ; Signal Transduction/physiology* ; RAW 264.7 Cells ; Mice, Knockout ; Mice, Inbred C57BL ; Male ; Flow Cytometry ; T-Cell Exhaustion